Methods for selective blood component removal.

The objective of therapeutic Extra Corporeal Blood Treatment (ECBT) is the removal or conversion of unwanted (mostly pathogenic) substances present in blood. At present ECBT is generally performed by using physical techniques such as dialysis, membrane filtration and centrifugation. These techniques are characterized by a low level of specificity leading to simultaneous removal of useful compounds. Administration of large quantities of substitution fluids (e.g., plasma-exchange, hemofiltration), is therefore often necessary. Higher levels of specificity can be obtained by using specific binders. Recent results obtained with synthetic polymers show the high potential of this technique. The use of biological substances, such as enzymes and antibodies, will enable an almost 100% level of specificity. It is obvious that the natural defence mechanism of the patient to be treated should not be triggered by the compounds used. This can be realized by either immobilizing the biological compounds on a solid phase, or by a physical separation between the compounds and the blood (e.g., by membranes). The former method seems more favorable since the efficiency will be optimal. The problems encountered, however, are numerous.

Preferential adsorption of high density lipoprotein from blood plasma onto biomaterial surfaces.

In the present study a two step enzyme immuno assay (EIA) was used for the investigation of the adsorption of proteins and lipoproteins from solutions and from blood plasma onto polymer surfaces. It was found that only a small adsorption of the major blood proteins occurred from plasma. Evidence is presented that the reason for this adsorption behaviour is a preferential adsorption of high density lipoprotein (HDL).

Rotavirus infections in calves in dairy herds.

Neonatal calf diarrhoea was studied in 115 calves of one dairy herd from January 1976 to June 1977. Two syndromes could be distinguished: a mild and short lasting 'early diarrhoea' within the first three days of life and a usually more severe 'late diarrhoea' from the fourth to the 14th day of life. The latter type of diarrhoea occurred almost exclusively during the first half of the year. Thirty-four out of 45 calves with late diarrhoea excreted rotaviruses, whereas only one of 34 calves with early diarrhoea excreted virus. In addition, rotavirus excretion was found in 11 calves that either remained healthy or had recovered from diarrhoea before virus was first detected. Similar findings were obtained in the spring of 1978 but in addition bovine coronavirus was recovered from four calves with late diarrhoea and from three healthy calves. One faecal sample obtained from a calf with diarrhoea on day 2 yielded K99+ Escherichia coli. Nearly all cows excreted rotavirus-specific antibodies in their colostrum but no relationship was found between the initial colostral antibody titre against rotavirus and the development of rotavirus-associated diarrhoea in the calf.

Rotavirus infections in calves: efficacy of oral vaccination in endemically infected herds.

A commercially available modified live reovirus-like (rotavirus) vaccine proved innocuous in calves deprived of colostrum and protected one out of three calves against challenge 72 h after vaccination. The vaccine was evaluated in two dairy herds in the 1977 calf season. No significant differences were observed in the incidence rates or severity of undifferentiated neonatal calf diarrhoea or rotavirus-associated late diarrhoea between calves given a placebo (76) and vaccinated (74) calves in these herds. Samples of colostrum contained specific antibodies against rotavirus and neutralisation of the vaccine virus by colostral antibody in the intestinal tract was considered the major reason for the failure of the vaccine to reduce the incidence of neonatal diarrhoea in these herds.

A simple method for the purification of 5-aminosalicylic acid. Application of the product as substrate in enzyme-linked immunosorbent assay (ELISA).

Commercially available 5-aminosalicylic acid (5-AS) was recrystallised in the presence of Na2S2O5. A completely colourless solution was obtained when the purified product was dissolved at a concentration of 1 mg/ml in a phosphate buffer containing EDTA and H2O2. No significant increase in absorption was found upon storage for 18 h at 4 degrees C. A 8-fold increase in sensitivity of an enzyme-linked immunosorbent assay for the detection of rotavirus antigens was demonstrated by using the modified substrate solution instead of the conventional substrate solution of crude 5-AS. In addition, P/N values did not significantly change between 2 and 18 h after addition of the substrate, thus rendering the time of reading less critical. No difference in sensitivity of the assay was found between modified 5-AS solution and ortho-phenylenediamine (OPD). However, since OPD requires special care in handling the modified 5-AS solution is preferred for use in routine ELISAs.

Detection of the K99 antigen of Escherichia coli in calf faeces by enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) is presented for the detection of the K99 antigen of Escherichia coli in calf faeces. False-positive reactions were not observed with K99-negative strains and with several viral antigens. Only bovine coronavirus caused slight positive reactions which could be eliminated by a blocking test. As compared with the conventional procedure for the detection of the K99 antigen, ELISA seemed to be a least as sensitive and had the advantage that samples could be stored at --20 degrees C before testing. In addition many samples could be handled at the same time and the results became available quickly. By carrying out the assay as a blocking test, specific antibody against K99 in serum or colostrum could be detected and titrated.

Detection of the K99 antigen of Escherichia coli in calf faeces by enzyme-linked immunosorbent assay (ELISA).

Summary An enzyme-linked immunosorbent assay (ELISA) is presented for the detection of the K99 antigen of Escherichia coli in calf faeces. False-positive reactions were not observed with K99-negative strains and with several viral antigens. Only bovine coronavirus caused slight positive reactions which could be eliminated by a blocking test. As compared with the conventional procedure for the detection of the K99 antigen, ELISA seemed to be at least as sensitive and had the advantage that samples could be stored at -20° C before testing. In addition many samples could be handled at the same time and the results became available quickly. By carrying out the assay as a blocking test, specific antibody against K99 in serum or colostrum could be detected and titrated.

Comparison of five diagnostic methods for the detection of rotavirus antigens in calf faeces.

Immunoelectroosmophoresis, complement fixation, immunofluorescence on inoculated cell cultures, electron microscopy, and an enzyme-linked immunosorbent assay were compared for the detection of rotavirus in faecal samples from calves. Rotavirus particles could be detected in 39 out of 98 faecal samples by electron microscopy. Immunofluorescence, complement fixation, and immunoelectroosmophoresis detected rotavirus antigens in 39, 42, and 30 samples, respectively. The enzyme-linked immunosorbent assay demonstrated rotavirus antigens in 49 faecal samples. The assay is not only sensitive but also simple to perform and suitable for large-scale testing.

The detection of rotavirus specific antibody in colostrum and milk by ELISA.

The blocking method of ELISA for the detection and titration of rotavirus-specific antibody in colostrum is described. The results obtained were positively correlated with those of a neutralizing antibody test. On one farm colostrum samples were obtained over a period of 18 months. No relationship was found between the titer of colostrum obtained shortly after calving, and the development of rotavirus-associated diarrhoea in calves. On a second farm only samples obtained during the calving season were tested. Within this restricted period high colostral antibody titers appeared to reduce the incidence of diarrhoea among calves and to delay the onset of rotavirus excretion in the faeces. These results are discussed in relation to the rapid decline in antibody content of colostrum after calving.

Enzyme-linked immunosorbent assay for diagnosis of rotavirus infections in calves.

An enzyme-linked immunosorbent assay for the diagnosis of rotavirus infection in calves is described. The assay was more efficient for the detection of rotavirus antigens in calf feces than were electron microscopy and immunoelectroosmorphoresis.

Rotavirus infections in calves. II. Experimental infections with a Dutch isolate.

Rotaviruses were observed electron microscopically in clarified faecal samples from calves with diarrhoea. Several herds with a history of scouring among neonatal calves were shown to be infected with rotaviruses. A bacteria-free faecal filtrate from a rotavirus positive sample administered orally induced diarrhoea in one out of two colostrum-deprived calves. Both calves excreted rotaviruses in their faeces and serum antibody responses were demonstrated by complement-fixation and indirect immunofluorescence tests and by immune electron microscopy. Purified rotavirus from faeces of the first calf induced profuse diarrhoea in three other colostrum-deprived calves, with subsequent shedding of the virus in the faeces. These calves also showed a serum antibody response. All calves recovered in one to seven days without treatment. Two colostrum-fed calves were removed from a rotavirus-affected dairy farm within 36 hours after birth and studied at the Institute. Both animals excreted rotaviruses and developed severe diarrhoea, although they had circulating maternal antibodies. Attempts to grow this rotavirus in cell cultures were not successful.

Identification and characterization of a papillomavirus from birds (Fringillidae).

From skin papillomas of the chaffinch (Fringilla coelebs), a virus has been purified and studied by physicochemical techniques and electron microscopy. The virions measure 52 nm in diameter and are composed of 72 morphological units arranged in a skew T = 7d surface lattice. A sedimentation coefficient of about 300S and a buoyant density of 1.34 g/ml in CsCl were determined for the particle. Its protein composition resembles that of human papillomavirus, and the circular double-stranded genome measures 2.6 micronm. This is the first demonstration of a member of the Papovaviridae family that affects a nonmammalian host.

The mechanism of replication of adenovirus type 5 DNA.

The analysis of replicating Ad5 DNA has led to a model for replication involving linear intermediates. The origin of replication has been localized at the molecular right end, suggesting the presence of a unique sequence of nucleotides which can be recognized by an initiation factor. On the other hand, the study of the molecular ends has shown, so far, a high degree of symmetry in the terminal nucleotide sequences of Ad5 DNA. A more detailed study of the nucleotide sequences at the ends is necessary to solve this problem.